The significant burden that results from dengue virus infection combined with the absence of effective vaccines or drugs makes the development of novel therapeutics a high priority. The process of viral entry is an attractive target for anti-dengue virus inhibitor development because interventions at this point of the viral life cycle can be used both to prevent and to treat infection;moreover, entry inhibitors would complement efforts directed towards development of more traditional viral protease and polymerase inhibitors. Currently, our understanding of the mechanisms of viral entry is insufficient to support rational design of viral entry inhibitors, and high-throughput assays that report on the fusogenic activity of the dengue virus envelope protein, which mediates viral entry, are sorely needed. The goal of this proposal is to develop and validate such assays for submission to the MLPCN. These assays should enable the discovery of inhibitors that block DENV entry at precise points along the pathway between pre- and postfusion conformations of the dengue virus envelope protein, which will provide much-needed tools for characterization of entry intermediates by structural and biochemical methods. This, in turn, will fuel efforts to develop dengue virus entry inhibitors by providing lead structures for optimization as well as by providing valuable mechanistic information to drive rational design efforts. In Aim 1, we propose to use develop a high-throughput fluorescence polarization assay to identify ligands of the dengue virus envelope protein that inhibit dengue virus infection. In Aim 2, we propose to develop a secondary assay that will be used to determine if primary screening hits inhibit dengue virus infection by blocking the fusion step of the entry process. Last, in Aim 3, we propose to validate our assay pipeline by screening a diverse 10,000- member compound library. We will use assays developed in Aims 1 and 2 as well as other assays previously developed in our laboratory to characterize the mechanisms of action of compounds with activity against dengue virus. The goal of this research is to validate assay methodology for eventual application in a high-throughput screening campaign against the MLPCN compound collection. A subsequent effort will focus on using these compounds as starting points for a medicinal chemistry campaign to develop optimized compounds that can be evaluated in preclinical and ultimately clinical DENV models and that can be used as mechanistic probes of dengue virus entry.